Important Guidelines for Transfection
- Form complexes using the amounts of DNA and Lipofectin® recommended. Optimize as necessary. Note: We recommend diluting DNA and Lipofectin® into Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) before complexing.
- Transfect cells at the confluence or cell density recommended. Optimize as necessary. Maintain the same seeding conditions between experiments.
- Do not add antibiotics to media during transfection as this causes cell death.
- For optimal results, perform transfection in medium without serum. Cells may be transfected in the presence of serum, if desired; however, complexes must be formed in serum-free medium.
- Test serum-free media for compatibility with Lipofectin® since some serumfree formulations (e.g. CD 293, 293 SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
Procedures are provided to transfect cells in a 6-well format (60-mm format for stable mammalian cell transfection). For other formats, vary the amounts of DNA, Lipofectin®, cells, and medium used in proportion to the relative surface area of the tissue culture vessel.
- One day before transfection, plate cells in growth medium without antibiotics such that they will be at the recommended confluence at the time of transfection.
For each transfection sample, prepare complexes as follows:
- a. Dilute 1-5 μg of DNA in 100 μl of Opti-MEM® I Reduced Serum Medium (or other medium) without serum.
- b. Mix Lipofectin® before use, then dilute 2-25 μl of Lipofectin® in 100 μl of Opti-MEM® I Medium (or other medium) without serum. Let stand at room temperature for 30-45 minutes.
- c. Combine the diluted DNA with diluted Lipofectin® (total volume = 200 μl). Mix gently and incubate for 10-15 minutes at room temperature (solution may appear cloudy).
- Add the 200 μl of complexes to cells. Mix gently by rocking the plate back and forth.
- Incubate cells at 37°C in a CO2 incubator for 5-24 hours.
- The following day, add 4 ml of complete growth medium to the cells.
- Incubate cells at 37°C in a CO2 incubator for 24-48 hours prior to testing for transgene expression.
|Condition||Cell no.||Growth med. vol.||Format||Confluence at txfn|
|Transient||1-2 x 105||2 ml||6-well||40-60%|
|Stable||1-2 x 105||4 ml||60-mm||30-50%|
Lipofectin® is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of BHK-21 cells with a reporter plasmid.
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